All organisms must regulate their genes precisely for normal development and to prevent disease states. However, significant gene copy number variation exists across genomes 1;therefore, coordinate regulation is required to equalize transcription levels 2 3. Our long-term goal is to describe the molecular mechanisms used to target genes for coordinate regulation, the essential initial step in their regulation. Dosage compensation is one of the best model systems for studying this process because all of the genes on a single chromosome are specifically identified and co-regulated. Drosophila, like mammals 4, increase the transcript levels of a large number of diversely regulated genes along the length of the single male X-chromosome precisely two-fold relative to each female X-chromosome 5. The objective of this application is to understand how dosage compensation in Drosophila is established, the critical first step in the regulatory process. The Drosophila Male Specific Lethal (MSL) complex is central to dosage compensation;it first identifies the X chromosome using a combination of cis-acting DNA sequences 6 and co-transcriptional recruitment by its roX (RNA on X) non-coding RNA components7 and then spreads into the bodies of active genes 8. However, we do not know how the MSL complex specifically identifies the MSL Recognition Element (MRE) sequences on the male X because known MSL components are insufficient for direct recognition of MREs in vitro 9. We used an innovative genetic screen for new regulators of dosage compensation that function in both males and females and thereby identified the essential CLAMP zinc-finger protein. Guided by strong preliminary data, we propose the following novel mechanism for identifying genes for coordinate regulation: CLAMP and the MSL complex associate inter-dependently, thereby generating a positive feedback amplification system that creates X- specificity from a two-fold X-enrichment of MRE sequences. The rationale for this work is that determining how the MSL complex specifically targets the X-chromosome will yield key insight into how genes are identified for coordinately regulation within sub-nuclear domains. We will test our novel mechanism using three specific aims: 1) We will define DNA sequence requirements for CLAMP binding in vivo and in vitro;2) We will identify CLAMP interacting proteins that mediate its interaction with the MSL complex. At the same time, we will define new interaction partners of a previously unstudied essential transcriptional regulator;3) We will establish the mechanism by which CLAMP and the MSL complex function inter-dependently at high affinity sites. Our proposed research is significant because we expect to describe for the first time the previously unknown mechanism that allows MSL complex to identify its high affinity sites, thereby defining the critical first step in establishing coordinate gene regulation. Defining the novel mechanism by which CLAMP and the MSL complex function together to generate a domain of enhanced transcription is likely to provide key insight into how genes are identified for coordinate regulation across species. PUBLIC HEALTH RELEVANCE: Our proposed research is relevant to public health because loss of precise regulation of genes underlies a large number of diseases including autism 10 and Chron's disease 11. Our research addresses the critical initial step in the coordinate regulation of genes, identifying target genes for subsequent regulation. Our findings are therefore relevant to the part of NIH's mission that pertains to developing fundamental knowledge that will provide tools for mitigating diseases.